Morphological characterization
Citrus endophytic strains were cultured on TSB and Corynebacterium-agar at 30oC, and yielded pale-yellow to orange-pink, circular colonies, while phytopathogenic strains presented yellow-ivory colonies. The cells of all evaluated strains were Gram-positive and rod shaped.
Diversity of endophytic Curtobacterium by AFLP technique
AFLP analysis was used as the main genome fingerprinting method to assess the overall genotypic similarity of the strains. The reproducibility of the results was tested at least once and less than 1.5% of the bands presented variation and were discarded from the present analysis. The analysis of endophytic Curtobacterium strains, C. luteum and subspecies of C. flaccumfaciens resulted in 147 bands for the three primer combinations, with 132 polymorphic bands (89.8%) which were used to determine genetic distances. The result of the comparative analysis of the AFLP profile of the citrus endophytic Curtobacterium strains, endophytic strains from other host plants, C. luteum, and C. flaccumfaciens reveled, at 50% similarity, that the evaluated isolates grouped into six clusters (Fig. 1). The phytopathogenic C. flaccumfaciens isolates clustered in a divergent group that shared only 36% of the bands with the other five clusters. The citrus endophytic Curtobacterium grouped into four clusters, which were separate from all other strains with similarity in < 29% of the bands.
In addition, the AFLP analysis, which included the C. flaccumfaciens pathovars, showed that the citrus endophytic strains grouped into different clusters and shared less than 35% similarity with these C. flacumfaciens pathovars, suggesting that these strains belong to another genotype. Previous studies using Acinetobacter species determined that a 50% similarity level was the threshold for the delineation of species by AFLP analysis (Nemec et al. 2001; Nemec et al. 2009). This AFLP analysis highlights the distinctness of the citrus strains from the other endophytic strains, C. luteum, and C. flaccumfaciens. This citrus population present 4 lineages (clusters) with high similarity in each cluster, suggesting that divergence is occurring in this bacterium and could be related to environmental selection. Representative citrus endophytic strains from the four clusters were further evaluated by 16S rRNA gene sequencing.
Phylogenetic analyses
The phylogenetic tree showed that all endophytic isolates belonged to the genus Curtobacterium within the family Microbacteriaceae. A high degree of 16S rRNA gene sequence similarity (≥ 98.3%) between all Curtobacterium species was observed with the 1030 bp sequence evaluated. The similarity between citrus endophytic strains ranged from 99.9 to 100%. The strains ER1/6T, ER1.4/2, SR4/1 and SR4/8 presented 100% similarity in this 16S rRNA gene sequence. The similarity of these endophytic strains and C. flaccumfaciens pathovars was ≥ 99.9% (Fig. S1). The same similarity level (≥ 99.9%) was observed between citrus endophytic Curtobacterium strains, and although the similarity between the evaluated species was extremely high, the 16S rRNA gene sequence analysis, with type strains of valid species for this Curtobacterium genus, grouped the endophytic strains in a different cluster (Fig. 2). This analysis showed that these endophytic strains, belonged to the lineage containing members of the genus Curtobacterium, as evidenced by the high (97%) bootstrap value.
The similarity between citrus endophytic strains and C. f. pathovars was ≤ 99.4%, while the similarity between C. f. pathovars and C. luteum was ≥ 99.9%; between C. f. pathovars and C. albidum, C. ammoniigenes, C. citreum, C. herbarum, and C. pusillum was ≥ 99.6%; and the similarity between C. f. pathovars and C. ginsengisoli was 98.6%. However, the similarity between citrus endophytic strains and the type strains was ≤ 99.7% for C. albidum, C. citreum, and C. pusillum, ≤ 99.3% for C. luteum, ≤ 99.1% for C. herbarum and C. ammoniigenes, and ≤ 98.4% for C. ginsengisoli. Although these citrus endophytic strains of Curtobacterium have high degrees of 16S rRNA gene sequence similarity to the established species of the genus, they clustered in a separate group (Fig. S1), indicating that they may be a divergent genotype in this genus.
In the phylogenetic tree based on 16S rRNA gene sequencing, strains ER1/6T, ER1.4/2, SR4/1 and SR4/8, which present 100% similarity in 16S rRNA sequence, grouped to the lineage containing members of the genus Curtobacterium as evidenced by the high bootstrap value, but the proximity to C. herbarum was not clear due to the low bootstrap value (Fig. 2). The DNA-DNA relatedness between Curtobacterium uspiensis sp. nov. ER1/6T and Curtobacterium flaccumfaciens pv. flaccumfaciens (LMG 3645T), Curtobacterium citreum (LMG8786T), Curtobacterium luteum (LMG8787T), Curtobacterium pusilum (LMG 8788T), and Curtobacterium herbarum (LMG19917T) was 35%, 48%, 42%, 37%, and 35%, respectively. The result for DNA-DNA hybridization between Curtobacterium flaccumfaciens pv. flaccumfaciens (LMG 3645T) and Curtobacterium herbarum (LMG19917T) was 33%, having been similar to results previously reported (Behrendt et al. 2002). This result support the proposal of a novel species within the genus Curtobacterium, for which the name Curtobacterium uspiensis sp. nov. is proposed with ER1/6T as the type strain.
Chemotaxonomic analysis
Major fatty acids of 11 citrus endophytic Curtobacterium strains belonging to different AFLP clusters are described in Table 2. The fatty acid profile of citrus endophytic isolates had similarities with C.f. pv. flaccumfaciens, C. f. pv. Poinsettiae, and C. f. pv. betae/oortii. The major cellular fatty acids of the strain ER1/6T were anteiso-C15:0 (48.01%), anteiso-C17:0 (32.68%), and iso-C16:0 (8.68%) as previously reported for members of the genera Curtobacterium and Rathayibacter (Kim et al. 2008). The strain ER1/6T contained menaquinones MK-9 as the predominant (85%) isoprenoid quinone with a smaller amount (14%) of MK-8, supporting the affiliation of these 11 strains, including ER1/6T, ER1.4/2, SR4/1 and SR4/8 to Curtobacteriumgenus.
Table 2
Fatty acid methyl ester analysis. Most similar Curtobacterium genotypes with citrus endophytic strains based on FAME-MIDI analysis, and the percentage of major fatty acids present in these strains.
Strains
|
Identity
|
Similarity
|
Fatty acids
|
C15:0 antiso
|
C17:0 antiso
|
C16:0 iso
|
AF2/5
|
C. f. pv. poinsettiae
|
0.763
|
42.96
|
33.81
|
8.89
|
PR2/2
|
C. f. pv. poinsettiae
|
0.834
|
45.05
|
30.97
|
10.70
|
AR1/2
|
C. f. pv. betae/ortii
|
0.813
|
45.54
|
29.25
|
10.45
|
SR1/6
|
C. f. pv. poinsettiae
|
0.776
|
46.41
|
29.19
|
9.74
|
SR4/1
|
C. f. pv. poinsettiae
|
0.891
|
47.92
|
30.48
|
7.33
|
SR4/8
|
C. f. pv. poinsettiae
|
0.887
|
48.03
|
30.54
|
7.32
|
ER1/5
|
C. f. pv. poinsettiae
|
0.871
|
45.18
|
32.68
|
8.68
|
ER1/6
|
C. f. pv. poinsettiae
|
0.898
|
48.01
|
30.64
|
7.48
|
ER1.4/2
|
C. f. pv. poinsettiae
|
0.897
|
48.02
|
30.62
|
7.45
|
EF1/6
|
C. f. pv. poinsettiae
|
0.764
|
50.88
|
17.69
|
12.12
|
SF2/17
|
C. f. pv. flaccumfaciens
|
0.750
|
45.86
|
33.53
|
9.39
|
Phenotypic characterizations
The differences in phenotypic characteristics between the proposed C. uspiensis and closely related type strains of Curtobacterium ammoniigenes B55T (Aizawa et al. 2007), C. citreum (Behrendt et al. 2002; Aizawa et al. 2007), C. pusillum (Behrendt et al. 2002; Aizawa et al. 2007), C. luteum (Behrendt et al. 2002; Aizawa et al. 2007), C. albidum (Behrendt et al. 2002; Aizawa et al. 2007), C. flaccumfaciens pv. flaccumfaciens (Behrendt et al. 2002; Aizawa et al. 2007), C. ginsengisoli (Kim et al. 2008), C. herbarum LMG 19917T (Behrendt et al. 2002)d plantarum (Dunleavy 1989) are listed in Table 3. Based on the full characterization of eleven C. uspiensis strains, this species characterized by gram-positive, rod-shaped cells that form orange/pink-colored colonies that grow optimally at 28oC. The enzymatic profiles of the endophytic strains were determined using a commercial system, API ZYM (API System, Bio Merieux) and analysis showed that these 11 endophytic strains are able to produce leucine arylamidase, α–galactosidase, β-galactosidase, α–glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, and α-manosidase. The endophytic strains produced variable results for esterase lipase (C8), cystine arylamidase, valine arylamidase, esterase (C4), and acid phosphatase, and they were unable to produce alkaline phosphatase, lipase (C14), trypsin, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase, β-glucuronidase, and α-fucosidase (Table 3).
Table 3
Physiological characteristics of C. uspiensis sp. nov. and closely related type strains of Curtobacterium species.
Characteristics a
|
Camb
|
Cit
|
Cpu
|
Clu
|
Cal
|
Cff
|
Cgi
|
Che
|
Cpl
|
ER1/6T
|
ER1.4/2
|
SR4/1
|
SR4/8
|
DNA G + C content (mol%)
|
68.8
|
72.1c
|
70.37 c
|
71.3c
|
ND
|
70.85c
|
65.8
|
71
|
76
|
72.2
|
ND
|
ND
|
ND
|
Colony color
|
Yellow
|
Yellow
|
Yellow
|
Yellow
|
Ivory
|
Yellow
|
Yellow
|
Orange
|
Yellow
|
Orange
|
Orange
|
Orange
|
Orange
|
Growth at 37°C
|
ND
|
W
|
W
|
W
|
W
|
W
|
ND
|
ND
|
W
|
W
|
W
|
W
|
W
|
Growth at 4oC
|
-
|
W
|
-
|
W
|
W
|
W
|
W
|
W
|
W
|
-
|
-
|
-
|
-
|
Motility
|
-
|
+
|
+
|
+
|
-
|
-
|
-
|
+
|
+
|
-
|
-
|
-
|
-
|
Hydrolysis of:
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Gelatin
|
-
|
-
|
+
|
-
|
+
|
+
|
ND
|
ND
|
+
|
-
|
-
|
-
|
-
|
L-Rhamnose
|
+
|
-
|
+
|
+
|
+
|
+
|
ND
|
ND
|
+
|
-
|
ND
|
ND
|
ND
|
N-Acetyl-D-glucosamine
|
-
|
-
|
+
|
+
|
-
|
W
|
-
|
ND
|
ND
|
+
|
ND
|
ND
|
ND
|
D-Trehalose
|
-
|
+
|
+
|
+
|
W
|
+
|
ND
|
ND
|
-
|
+
|
+
|
+
|
+
|
Assimilation of:
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Tween 40
|
-
|
W
|
-
|
W
|
+
|
W
|
ND
|
ND
|
ND
|
+
|
+
|
+
|
+
|
N-Acetyl-D-galactosamine
|
-
|
-
|
-
|
-
|
-
|
+
|
ND
|
ND
|
ND
|
-
|
ND
|
ND
|
ND
|
N-Acetyl-D-glucosamine
|
-
|
+
|
-
|
+
|
W
|
+
|
ND
|
ND
|
ND
|
+
|
ND
|
ND
|
ND
|
D-Arabitol
|
-
|
-
|
W
|
-
|
-
|
+
|
ND
|
ND
|
ND
|
-
|
ND
|
ND
|
ND
|
D-Cellobiose
|
-
|
+
|
-
|
+
|
+
|
+
|
ND
|
ND
|
-
|
+
|
+
|
+
|
+
|
L-Fucose
|
-
|
+
|
-
|
+
|
-
|
W
|
-
|
ND
|
ND
|
-
|
-
|
-
|
-
|
Gentiobiose
|
-
|
-
|
+
|
-
|
+
|
+
|
ND
|
ND
|
ND
|
+
|
ND
|
ND
|
ND
|
myo-Inositol
|
-
|
-
|
-
|
-
|
-
|
+
|
ND
|
ND
|
+
|
+
|
+
|
+
|
+
|
a-D-Lactose
|
+
|
+
|
-
|
+
|
+
|
+
|
ND
|
ND
|
-
|
+
|
-
|
+
|
-
|
D-Mannitol
|
+
|
-
|
+
|
-
|
W
|
+
|
-
|
+
|
d
|
+
|
+
|
+
|
+
|
D-Melibiose
|
+
|
+
|
-
|
+
|
W
|
+
|
-
|
+
|
ND
|
+
|
ND
|
ND
|
ND
|
Methyl b-D-glucoside
|
-
|
-
|
+
|
-
|
W
|
+
|
ND
|
ND
|
ND
|
+
|
ND
|
ND
|
ND
|
D-Raffinose
|
-
|
-
|
-
|
-
|
+
|
+
|
ND
|
ND
|
-
|
-
|
ND
|
ND
|
ND
|
D-Sorbitol
|
+
|
-
|
+
|
-
|
+
|
+
|
-
|
+
|
ND
|
+
|
+
|
+
|
+
|
Turanose
|
+
|
-
|
+
|
-
|
+
|
+
|
ND
|
ND
|
ND
|
+
|
ND
|
ND
|
ND
|
D-Gluconic acid
|
+
|
-
|
+
|
-
|
+
|
+
|
ND
|
ND
|
+
|
+
|
ND
|
ND
|
ND
|
D-Glucuronic acid
|
-
|
-
|
-
|
-
|
-
|
+
|
+
|
+
|
ND
|
-
|
ND
|
ND
|
ND
|
a-Ketobutyric acid
|
-
|
W
|
-
|
+
|
-
|
-
|
-
|
+
|
ND
|
-
|
ND
|
ND
|
ND
|
DL-Lactic acid
|
+
|
W
|
-
|
W
|
-
|
+
|
ND
|
ND
|
+
|
-
|
-
|
-
|
-
|
Bromosuccinic acid
|
-
|
W
|
-
|
-
|
-
|
+
|
ND
|
ND
|
ND
|
-
|
ND
|
ND
|
ND
|
L-Alanine
|
-
|
-
|
-
|
-
|
W
|
+
|
-
|
ND
|
ND
|
-
|
-
|
-
|
-
|
L-Glutamic acid
|
-
|
+
|
-
|
-
|
-
|
+
|
ND
|
ND
|
ND
|
+
|
+
|
+
|
+
|
Lactate
|
+
|
W
|
-
|
W
|
-
|
+
|
-
|
ND
|
|
+
|
+
|
+
|
+
|
Acetic acid
|
ND
|
+
|
+
|
+
|
+
|
+
|
ND
|
ND
|
+
|
+
|
+
|
+
|
+
|
Malic acid
|
ND
|
+
|
-
|
+
|
-
|
+
|
ND
|
ND
|
d
|
-
|
-
|
-
|
-
|
a-Ketoglutaric acid
|
ND
|
+
|
-
|
-
|
-
|
D
|
ND
|
ND
|
+
|
-
|
ND
|
ND
|
ND
|
Citric acid
|
ND
|
+
|
-
|
-
|
+
|
+
|
ND
|
ND
|
d
|
-
|
-
|
-
|
-
|
Propionic acid
|
ND
|
-
|
+
|
-
|
-
|
-
|
ND
|
ND
|
+
|
-
|
-
|
-
|
-
|
Arginine
|
ND
|
-
|
-
|
-
|
-
|
-
|
ND
|
ND
|
-
|
-
|
-
|
-
|
-
|
Glucose
|
ND
|
W
|
W
|
+
|
W
|
W
|
ND
|
ND
|
+
|
+
|
+
|
+
|
+
|
Fructose
|
ND
|
W
|
W
|
+
|
W
|
W
|
ND
|
ND
|
+
|
+
|
+
|
+
|
+
|
Mannose
|
ND
|
W
|
+
|
+
|
-
|
W
|
ND
|
ND
|
+
|
+
|
+
|
+
|
+
|
Galactose
|
ND
|
W
|
+
|
-
|
-
|
-
|
ND
|
ND
|
+
|
+
|
+
|
+
|
+
|
Sucrose
|
ND
|
-
|
-
|
-
|
-
|
-
|
ND
|
ND
|
-
|
+
|
+
|
+
|
+
|
Maltose
|
ND
|
W
|
W
|
-
|
W
|
W
|
ND
|
ND
|
+
|
+
|
+
|
+
|
+
|
Glycerol
|
ND
|
-
|
-
|
-
|
-
|
-
|
ND
|
ND
|
-
|
+
|
+
|
+
|
+
|
aCam: Curtobacterium ammoniigenes B55T (5); Cit: C. citreum (3, 5); Cpu: C. pusillum (3, 5); Clu: C. luteum (3, 5); Cal: C. albidum (3, 5); Cff: C. flaccumfaciens pv. flaccumfaciens (3, 5); Cgi: C. ginsengisoli (6); Che: C. herbarum LMG 19917T (3); Cpl: C. plantarum (41). |
bND: not determined; - : no growth; +: strong growth observed; W: weak growth, d: 11–89% of the strains were positive |
cSequenced genomes deposited at GenBank (NCBI). |
Genome analysis and secondary metabolic gene clusters
Based on genome sequencing, the DNA G + C content of the type strain is 72.2%. The endophytic bacterium C. uspiensis ER1/6T reduces symptoms caused by Xylella fastidiosa in Catharanthus roseus (Lacava et al. 2007) and in vitro (Lacava et al. 2004), and produces, based on ESI-MS/MS fragmentation profile, phospholipids including the classes of glycerophosphocholine, glycerophosphoglycerol, and glycerophosphoinositol as well as several fatty acids (Araújo et al. 2018). Therefore, we screened the genome sequence for the presence of genes encoding for the biosynthesis of phospholipids, antibiotics, and other metabolites contributing to plant disease suppression. We detected three clusters encoding secondary metabolites (Table 4), including a bacteriocin similar to cell wall-active bacteriocin lactococcin 972 like (Martínez et al. 2008), the putative pathway for the desferrioxamines (Fig. S2 and S3), a siderophore belonging to hydroxamate group (Ronan et al. 2018), and the rare C50 carotenoid decaprenoxanthin (Fig. S4 and S5), a pigment used for coloration of food, feed and beverages (Henke et al. 2017).
Table 4
Identified clusters of secondary metabolites in Curtobacterium uspiensis ER1/6T genome (NZ_MJAK01000000.1).
Genbank acession
|
Location
|
Identified cluster of secondary metabolite
|
NZ_MJAK01000001.1
|
22,753 − 28,012 nt
|
siderophore Desferrioxamine-like
|
NZ_MJAK01000003.1
|
220,629 − 226,575 nt
|
terpene C50 carotenoid-like
|
NZ_MJAK01000008.1
|
69,090 − 69,623 nt
|
bacteriocin Lactococcin 972 like
|
Table 5. Description of Curtobacterium uspiensis sp. nov. according to Digital Protologue TA00526 assigned by the www.imedea.uib.es/dprotologue website.
Taxonumber
|
TA00526
|
Species name
|
Curtobacterium uspiensis
|
Genus name
|
Curtobacterium
|
Specific epithet
|
uspiensis
|
Species status
|
sp. nov.
|
Species etymology
|
us.pi.en’sis. N.L. n. uspiensis from USP, named in reference to USP – University of São Paulo - where this bacterium has been studied
|
Authors
|
Araújo WL, Belmonte UCF, Dourado MN, Garrido LM, Yara R, Azevedo, JL
|
Title
|
Curtobacterium uspiensis sp. nov., an endophytic bacterium isolated from Citrus sinensis (sweet orange) in Brazil
|
Corresponding author
|
Welington Luiz Araújo
|
E-mail of the corresponding author
|
[email protected]
|
Submitter
|
Welington Luiz Araújo
|
E-mail of the submitter
|
[email protected]
|
Designation of the type strain
|
ER1/6
|
Strain collection numbers
|
|
16S rRNA gene accession number
|
|
Genome accession number [RefSeq]
|
MJAK00000000
|
Genome status
|
Draft
|
Genome size
|
3,368.952 kbp
|
GC mol%
|
72.2
|
Country of origin
|
Brazil
|
Region of origin
|
São Paulo
|
Date of isolation
|
1997
|
Source of isolation
|
Citrus sinensis branch
|
Sampling date
|
1997
|
Geographic location
|
Novais City
|
Number of strains in study
|
20
|
Source of isolation of non-type strains
|
Citrus branches
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Growth medium, incubation conditions [temperature, pH, and further information] used for standard cultivation
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Tryptic Soy (TS) agar plates at 28oC under aerobic conditions.
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Alternative medium 1
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Corynebacterium - agar (10 g Casein peptone, tryptic digest; 10 g Yeast extract; 5 g glucose; 5 g NaCl in 1000 mL of water) at 28oC under aerobic conditions
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Conditions of preservation
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stored at -80oC in glycerol 20% and lyophilized.
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Gram stain
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Positive
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Cell shape
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Rod
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Motility
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Non-motile
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Colony morphology
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pale yellow to orange-pink with irregular borders and smooth surface
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Temperature optimum
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25 to 30
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pH optimum
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6,8
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pH category
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Neutrophile
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Salinity optimum
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<1
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Salinity category
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halotolerant
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Relationship to O2
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Aerobe
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O2 conditions for strain testing
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Aerobiosis
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Carbon source used [class of compounds]
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Sugars, amino acids
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Catalase
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Positive
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Positive tests with BIOLOG (BIOP)
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Tween 40, N-acetyl-D-glucosamine, D-cellobiose, gentiobiose, D-lactose, D-mannitol, D-melibiose, methyl β-D-glucoside, D-sorbitol, turanose, D-gluconic acid, L-glutamic acid, acetic acid, lactate, D-fructose, D-glucose, D-galactose, D-mannose, maltose, sucrose, glycerol,
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Negative tests with BIOLO (BION)
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N-Acetyl-D-galactosamine, D-arabitol, L-fucose, myo-inositol, D-raffinose, D-glucuronic acid, α-ketobutyric acid, DL-lactic acid, bromosuccinic acid, L-alanine, malic acid, α-ketoglutaric acid, citric acid, propionic acid and arginine. The type strain was negative for gelatin hydrolysis
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Positive tests with API (APIP)
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leucine arylamidase α–galactosidase, β-galactosidase, α–glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-manosidase
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Negative tests with API (APIN)
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alkaline phosphatase, lipase (C14), trypsin, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase, β-glucuronidase, α-fucosidase
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Variable tests with API (APIV)
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esterase lipase (C8), cystine arylamidase, valine arylamidase, esterase (C4), acid phosphatase
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Quinone type (QUIN)
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MK-9 (85%), MK-8 (14%)
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Major fatty acids (FAME)
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anteiso-C15:0 (47.28%), anteiso-C17:0 (32.45%), iso-C16:0 (9.37%)
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Biosafety level
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1
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Habitat
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portion of plant tissues
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Biotic relationship
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Endophyte
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Symbiosis with the host
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Citrus plants
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Known pathogenicity
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None
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